Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
Background/Disease Information:
Three β-carbonic anhydrases are present in the human pathogen Mycobacterium tuberculosis. Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a beta-carbonicanhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a beta-carbonicanhydrase can switch between two states. There is a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium. Essentiality of this protein: Gene/Ortholog: mtu1305 (OG4_19505); Phenotype: essential; Source study: nmpdr
Complex of proteins?: No Druggable Target: It's known to be druggable by orthology.
Though enzyme activity can be measured by CO2 hydration, the surrogate enzyme assay using 4-nitrophenyl acetate, measuring the amount of 4-nitrophenol cleaved.
-- Cost and quantity of substrate reagents and supplier:N8130-10G $48.20
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1YLK
-- For Homology Model option: methanoarchaen Methanobacterium thermoautotrophicum (Cab).
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- % Positives---- Max % Identities:
---- Chain used for homology:
Figure 1: PyMol representation of the protein target, β-carbonic anhydrases, displayed as cartoon with the ligand, SCN shown as sticks. Current Inhibitors: 2-amino-pyrimidin-4-yl-sulfanilamide, 2-aminobenzenesulfonamide, 3-(4-sulfamoylphenyl)propanoic acid, 3-bromosulfanilamide, 4,5-dichloro-benzene-1,3-disulfonamide.
Expression Information (has it been expressed in bacterial cells): expressed in Escherichia coli Purification Method: glutathione Sepharose 4B column chromatography and sulfonamide affinity chromatography Image of protein (PyMol with features delineated and shown separately):
GC% Content for gene: 55% CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
(http://tdrtargets.org/targets/view?gene_id=7856)
NCBI Gene # or RefSeq#: GI:78101214
Protein ID: 1YLK
Organism (including strain): Mycobacterium tuberculosis H37Rv
Etiologic Risk Group (see link below):
Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
Background/Disease Information:Three β-carbonic anhydrases are present in the human pathogen Mycobacterium tuberculosis. Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a beta-carbonicanhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a beta-carbonicanhydrase can switch between two states. There is a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium.
Essentiality of this protein:
Gene/Ortholog: mtu1305 (OG4_19505); Phenotype: essential; Source study: nmpdr
Complex of proteins?: No
Druggable Target: It's known to be druggable by orthology.
EC#: 4.2.1.1
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=4.2.1.1
Screenshot of BRENDA enzyme mechanism schematic:
Enzyme Assay information:
-- link to assay or assay reagents (substrates):
http://www.sigmaaldrich.com/img/assets/18220/Carbonic_Anhydrase_-_CSRD.pdf
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/n8130pis.pdf
Though enzyme activity can be measured by CO2 hydration, the surrogate enzyme assay using 4-nitrophenyl acetate, measuring the amount of 4-nitrophenol cleaved.
-- link (or citation) to paper that contains assay information
http://pubs.acs.org/doi/pdf/10.1021/jm9000488
-- Cost and quantity of substrate reagents and supplier: N8130-10G $48.20
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1YLK
-- For Homology Model option: methanoarchaen Methanobacterium thermoautotrophicum (Cab).
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- % Positives---- Max % Identities:
---- Chain used for homology:
Figure 1: PyMol representation of the protein target, β-carbonic anhydrases, displayed as cartoon with the ligand, SCN shown as sticks.
Current Inhibitors:
2-amino-pyrimidin-4-yl-sulfanilamide, 2-aminobenzenesulfonamide, 3-(4-sulfamoylphenyl)propanoic acid, 3-bromosulfanilamide,
4,5-dichloro-benzene-1,3-disulfonamide.
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=4.2.1.1&Suchword=&organism%5B%5D=Mycobacterium+tuberculosis&show_tm=0
Expression Information (has it been expressed in bacterial cells):
expressed in Escherichia coli
Purification Method:
glutathione Sepharose 4B column chromatography and sulfonamide affinity chromatography
Image of protein (PyMol with features delineated and shown separately):
Amino Acid Sequence:
ATGACCGTTACCGATGACTACCTGGCGAATAACGTTGACTACGCGTCTGGTTTCAAAGGT
CCGCTGCCGATGCCGCCGTCTAAACACATCGCAATCGTTGCGTGCATGGACGCACGTCTG
GACGTTTACCGCATGCTGGGTATCAAAGAAGGTGAAGCGCATGTCATCCGCAACGCGGGT
TGCGTTGTAACCGACGACGTTATCCGTTCTCTGGCGATCTCTCAGCGTCTGCTCGGTACC
CGTGAGATCATCCTGCTGCACCACACCGACTGCGGTATGCTGACCTTCACGGACGACGAC
TTCAAACGTGCCATCCAGGACGAAACTGGTATCCGTCCGACCTGGTCTCCGGAATCTTAC
CCGGACGCGGTTGAAGACGTTCGTCAGAGCCTGCGCCGTATCGAAGTTAACCCGTTCGTT
ACCAAACACACCTCTCTGCGTGGTTTCGTTTTCGACGTTGCGACCGGCAAACTGAACGAG
GTTACCCCGTAA
Length of protein: 172 amino acids; 516 base pairs
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
GC% Content for gene: 55%
CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):